Antisense oligonucleotides are synthetic oligonucleotides which are designed to bind to RNA by Watson-Crick base paring. This binding, or hybridization can result in the selective inhibition of RNA expression. Additionally, the antisense oligonucleotides may block gene expression by inhibition of replication or transcription of DNA by Hoogsteen bonding. This type of bonding results in the formation of a triple helix wherein the oligonucleoide analog binds in a sequence-specific manner in the major groove of the DNA duplex structure. A number of cellular processes can be inhibited depending on where the oligonucleotide hybridizes on regions of DNA or mRNA. To be effective as therapeutic agents, oligonucleotides must reach the interior of target cells unaltered. This requires the oligonucleotides to be able to penetrate the cell membrane and to be resistant to intra- and extracellular nucleases. Additionally, antisense oligonucleotides can be used for diagnostic purposes by coupling the oligonucleotide to a suitable imaging agent (i.e., radiolabel, fluorescent tag, or biotin) or solid support.
Natural oligonucleotides are negatively charged and do not easily penetrate the cell membrane. Additionally, they are susceptible to degradation by nucleases which cleave the phosphodiester linkage. For these reasons, efforts to prepare pharmaceutically active oligonucleotides have focused on synthetic analogs which address these problems.
A number of strategies have been employed in the preparation of antisense oligonucleotides including, replacement of one non-bridging oxygen in phosphodiester linkages with sulfur (see, Cohen, et al., U.S. Pat. Nos. 5,264,423 and 5,276,019); replacement of both non-bridging oxygens in the phosphodiester linkages with sulfur (see, Brill, et al., J. Am. Chem. Soc. 111:2321 (1989)); use of nonionic alkyl and aryl phosphonates in place of the phosphate linking group (see, Miller, et al., U.S. Pat. Nos. 4,469,863 and 4,757,055); and, replacement of phosphorus atoms with carbon or silicon (see, Stirchak, et al., J. Org. Chem. 52:4202-4206 (1987) and Cormier, et al., Nucleic Acids Res. 16:4583 (1988), respectively). These strategies and related efforts are the subject of a recent review (see, Uhlmann, et al., Chem. Reviews 90:543-584 (1990)).
More recently, others have focused on the preparation of charge-neutral antisense oligonucleotides which have zwitterionic moieties attached to the phosphodiester backbone (see, Cook, WO 93/15742 (1993)).
Base modification has been virtually ignored as a method by which to engineer oligonucleotides for use as antisense agents. However, nature has employed a design strategy of attaching a positively charged species to a thymidine base. Thus, bacteriophage .phi.W-14 is known to replace approximately half of the thymines present in its DNA with positively charged .alpha.-putrescinylthymine. This results in approximately one hypermodified base every eight nucleotides on average. These and other hypermodified bacteriophage DNAs have been shown to resist a variety of endonucleases and some exonucleases as well. While it is known from these and other examples that the DNA major groove will tolerate some substitution without a significant deleterious effect on duplex formation, little information is available about the effect of introducing contiguous, bulky, charged modifying groups into the major groove.